DNA Collection Sticker and Method for Isolating DNA From the Sticker

ABSTRACT

The present invention relates a sticker for DNA collection and a method for isolating DNAs using the same. Particularly, the sticker for DNA collection is covered with a paint solution comprising EDTA, Tris, SDS and peyonine to isolate keratins exclusively when attached onto human skin and detached. Further, the specific sticker for DNA collection separates DNAs efficiently to amplify genes by using a PCR technique. Therefore, the present invention can be applied to identify a real child and investigate a crime with a fingerprint and to screen genetic diseases.

TECHNICAL FIELD

The present invention relates to a sticker for DNA collection and amethod for extracting DNAs using the same, more particularly to a DNAcollection sticker, which conveniently separates keratin attached tohuman skin without blood or hair to collect a human gene and amplify thegene by using a PCR technique for identifying a real child andinvestigating a crime, and a method for collecting DNAs efficientlyusing the same.

BACKGROUND ART

Generally, DNA (deoxyribonucleic acid), a molecule containing geneticinformation in a human body is stored in 23 pairs of human chromosomes.In respect of genetic information, DNA is composed of exon encoding aprotein and intron, a non-coding region. The intron region, especiallyAlu sequence has a repeated pattern specific for an individual,depending upon its genetic lineage. For this reason, it is being adoptedfor a gene fingerprint to screen a similar or same sequence scatteredonto a genome, i.e. microsatellite or VNTR (Variable Number of TandemArray).

Presently, in order to collect DNAs from a human body, the method usingblood or hair has been disclosed. However, there are several problems.This technique is complicated in the procedure as well as oftendiscomforted.

Further, the sticker for DNA collection has been developed to exploitskin keratin easily fallen apart from a human body. The conventionalsticker is problematic, because the keratin containing DNAs is toodifficult to be displaced exclusively from the adhesive ingredient onthe sticker. In detail, the adhesive may agglomerate with DNAs under anormal condition when separating DNA, because the effective ingredientof adhesive is a polymer like DNA.

Therefore, the present inventors have demonstrated the sticker that isprepared by coating a paint solution comprising EDTA (ethylene diaminetetraacetate), Tris and SDS (sodium dodecyl sulphate) in the KoreanPatent Application NO. 2000-74853 (hereinafter, referred to as“conventional invention”). The resulting sticker can collect DNAsexclusively from skin keratin in a human body. Then, the DNA resultantis separated from the sticker by using a phenol extraction method.Unfortunately, this process has not improved the DNA yieldsatisfactorily, compared to conventional techniques, even thoughexploiting the sticker and the phenol extraction.

DISCLOSURE Technical Problem

In order to settle above-mentioned problems, the present inventors havedeveloped the following invention successfully. The object of thepresent invention is to provide a sticker for DNA collection thatimproves the productive yield of DNA.

The other object of the present invention is to provide a method forpurifying DNA efficiently using the sticker for DNA collection, whichimproves to conveniently separate keratin attached from human skin toobtain a human gene.

Technical Solution

Precisely, the present invention comprises several steps as follows: (1)preparing a sticker; (2) collecting keratin from human skin; (3)purifying DNAs; (4) amplifying DNA by performing PCR; and (4)identifying various genes and genetic information of individual.

In order to achieve the objects of the present invention, the stickerprepared by coating a paint solution comprising EDTA (ethylene diaminetetraacetate), Tris and SDS (sodium dodecyl sulphate) and Peyonine isattached and detached, before collecting to analyze genes from a humanbody. Then, the resulting sticker is reacted with the paint solution at30˜45° C. for 40˜80 minutes. After that, proteinase K is added andreacted at 40˜68° C. for 30˜100 minutes.

The method for separating DNA using the sticker for DNA collection ofthe present invention has a feature to comprise following steps:

i) coating a paint solution comprising 0.05˜1 mol of EDTA (pH 8.0),0.002˜0.015 mol of Tris (pH 8.0), 30˜40 vol % of SDS (approximately1˜1.4 mol) and 3% Peyonine onto one side of a commercial sticker forhuman use to prepare a sticker for DNA collection;

ii) attaching the sticker onto human skin and detaching to collect humankeratin;

iii) adding a protein precipitation reagent to purify DNA from theresulting keratin;

iv) performing PCR to amplify the resulting DNA;

v) examining the PCR product to identify various genes and geneticinformation of individual within a DNA specimen.

Hereinafter, the present invention will be described more clearlyaccording to following steps.

Step 1 Preparation of Sticker for DNA Collection

The sticker used in the present invention is a product for human usecontaining a nontoxic adhesive ingredient and commercially available.The commercial sticker is coated with a paint solution comprising EDTA(ethylene diamine tetraacetate), Tris and SDS (sodium dodecyl sulphate)and Peyonine onto one side containing an adhesive agent to manufacturethe sticker for DNA collection.

The paint dose should be adjusted properly. If higher, the sticker forDNA collection is too difficult to attach onto a human body. Incontrast, if lower, it is too hard to separate human keratin fromadhesive agent. Preferably, the surface for attachment is coated with apaint solution in 70˜85% and more preferably, in 75%. Also, the paintthickness should be controlled properly to contact an adhesive agent anda human body. Preferably, the paint thickness is less than 3 mm.

In the paint solution, EDTA plays a role to remove a magnesium ion andweaken the cell membrane because the magnesium is essential to maintainthe overall structure of cell membrane; and further, help intact DNAscollected by inhibiting intracellular enzymes. In addition, Tris, abuffering system is nontoxic, cheap and suitable for frequent use. SDS,a sort of detergent plays a role to help a cell lysis by removing lipidmolecules and cause the dissociation of cell membrane. In order toprepare the paint solution working properly, 0.05˜1 mol of EDTA (pH 8.0)and 5˜15 mol of Tris (pH 8.0) are mixed with 3% peyonine and SDSadjusting to 30˜40 vol % to reach 7,000˜8,0001 ml of total volume byadding distilled water. Preferably, 0.1 mol of EDTA and 10 mols of Trisare mixed with 3% peyonine and SDS adjusting to 37.5 vol % to reach7,500 ml of total volume by adding distilled water.

Step 2 Collection of Human Keratin

The sticker of the present invention prepared above can be attached ontoany region in a human body. Preferably, the sticker can be applied ontoa keratinous region such as elbow, axilla, arm, ankle and face. Thesticker of the present invention may not affected by a duration period.Right after the attachment, the sticker of the present invention cancollect the specimen in a proper amount.

Step 3 Purification of DNAs

From the sticker prepared above, human keratins are collected and then,DNAs are purified as follows.

(i) Above all, an adhesive portion is cut to a proper size(approximately 4 cm×6 cm) from the sticker and immersed in DNAextraction solution having the same composition and ratio with that ofthe paint solution. This composition and ratio facilitates thedissociation between adhesive ingredients of the sticker and humankeratins collected. Thus, the paint solution is dissolved easily towardDNA extraction solution.

The resulting paint solution is incubated under an immersed state at 30°C. ˜45° C., preferably 30° C. for 40˜80 minutes and more preferably, 37°C. for 60 minutes. If incubated at under 30° C., the dissolution ratioof the paint solution decreases. The paint solution between adhesiveingredients of the sticker and human keratins collected is lessrecovered due to low activities of the DNA extraction solution and thepaint solution. As a result, the recovery ratio of DNA decreases to lessthan 60% and further analyses could not be accomplished. In contrast, ifincubated at over 45° C., the adhesive ingredient, polymer substance maybe dissolved into the DNA extraction solution from the sticker anddecrease the activities of the DNA extraction solution and the paintsolution. As a result, the recovery ratio of the adhesive ingredientincreases remarkably, compared to that of the human keratins and furtherDNA analyses as well as PCR amplification could not be accomplished.

In the meantime, if incubated for under 40 minutes, the dissolutionperiod is too short to separate adhesive ingredients of the sticker andhuman keratins fully. Further DNA analyses and PCR amplification couldbe hardly accomplished. If incubated for over 80 minutes, the adhesiveingredients of the sticker are dissociated together during a finalalcohol extraction in the DNA purification. Further PCR amplificationcould be hardly accomplished. Accordingly, the incubation should beperformed under a proper condition.

(ii) After the incubation, proteinase K, a protein degradation enzyme isadded to remove proteins. At this moment, proteinase K is reacted at40˜68° C. for 30˜100 minutes and preferably, at 5° C. for 90 minutes. Ifreacted for over 100 minutes, the recovery amount of DNA decreases dueto the action of Dnase after separated together from skin. If reactedfor under 30 minutes, proteins are not discarded sufficiently. Incontrast, if reacted at under 40° C. or over 68° C., the activity ofproteinase K, a protein degradation enzyme sensitive to temperaturedecreases. The keratins collected form a human body remainsagglomerated. Further, the recovery ratio of DNA decreases. Especially,if reacted at over 68° C., DNAs separated above becomes denatured.

(iii) Protein precipitation solution is added in a half volume of theresulting solution and centrifuged to precipitate proteins. The waterlayer placed in supernatant is separated by using a pipette. After that,isopropanol is added to the resultant in the same volume and againcentrifuged to obtain DNA specimen. Then, 70% ethanol is poured to theresulting specimen in the same volume under a salt like sodium ion andcentrifuged at −20° C. to precipitate DNAs (DNA concentration).

Step 4 Amplification of DNA by PCR (Polymerase Chain Reaction)

The resulting DNAs is washed by using 70% ethanol, dried and dissolvedwith TE buffer. The purified DNA can be amplified for a short time byperforming PCR (polymerase chain reaction). Also, the DNA collected fromthe sticker can be amplified by performing the PCR. Through thisprocedure, DNAs is produced in a sufficient amount even after 2˜3 cyclesof PCR.

Step 5 Identification of Various Genes and Genetic Information ofIndividual

The DNA specimen prepared above can be applied to screen various genesand genetic information of individual. Further, the geneticcharacteristics can be used to identify a real child or investigate asuspected person in a crime. In addition, the DNA collected by thisprocedure can be utilized to screen genetic diseases as well as toscreen such a fingerprint. In detail, Southern blotting and DNA chipusing the principle of hybridization between DNAs are accomplished inorder to identify various genes and genetic information of individual.

As illustrated above, the sticker for DNA collection and the method forcollecting DNA using the same enables keratin attached from human skinseparated easily without blood or hair and further human gene separatedconveniently and amplified by using a PCR technique. Therefore, thepresent invention may attain the effect that identifies a real child,investigates a crime and the like.

That is to say, the specific sticker for DNA collection is attached ontoskin, detached and applied to separate DNAs efficiently, whichfacilitates the identification of personal genetic information.

ADVANTAGEOUS EFFECTS

As described above, the sticker for DNA collection and the method forcollecting DNA using the same according to the present invention isadvantageous in that, enable keratin attached from human skin separatedeasily without blood or hair. Further, the present invention enables ahuman gene separated conveniently and amplified by using a PCRtechnique. Therefore, the present invention may attain the effect thatidentifies a real child, investigates a crime and the like. Hence, thesticker for DNA collection and the method for collecting DNA using thesame are industrially useful in genetic engineering fields.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 depicts the schematic diagram of the procedure separating DNAsfrom a sticker.

FIG. 2 depicts the comparison of productive yields of PCR productsprepared by DNAs from the sticker of the present invention andconventional stickers.

FIG. 3 depicts the comparison of PCR products prepared by the DNAseparated with a reagent precipitating proteins and through a phenolextraction from the sticker of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Practical and presently preferred embodiments of the present inventionare illustrated as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of Sticker and Collection of Human Keratin

In order to prepare a paint solution, 0.1 mol of EDTA (pH 8.0) and 10mol of Tris (pH 8.0) were mixed with 3% peyonine and SDS adjusting to30˜40 vol %. Then, the final volume was adjusted to 7,500 ml by addingdistilled water. The resulting solution was coated onto one side ofstickers for human use that contains an adhesive ingredient nontoxic toa human body to cover 75% of the side area in 2 mm of thickness.

The sticker coated above was attached onto the palm and after 10minutes, detached again.

Mode of the Invention Example 2 Purification of DNAs from Human KeratinAttached onto Sticker

The sticker prepared in Example 1 was cut to 4 cm×6 cm of square,submerged in a paint solution having the same composition and ratio withthose of Example 1 and reacted at 37° C. for 60 minutes. After that,proteinase K, a protein degradation enzyme was added and incubated at65° C. for 90 minutes.

Then, a protein precipitation solution was added in a half volume of theresulting solution and centrifuged to discard proteins. The water layerplaced in supernatant was separated by using only a pipette. After that,isopropanol was added to the resultant in the same volume and againcentrifuged to obtain DNA specimen. Then, 70% ethanol was poured to theresulting specimen in the same volume under a salt like sodium ion andcentrifuged at −20° C. to precipitate DNAs (DNA concentration).

Experimental Example 1 Examination of DNA Amounts Under VariousConditions

The stickers prepared in Example 1 and 2 were analyzed under variousconditions, according to the composition of adhesive agent, thetemperature and time period of incubation and the reaction period andtemperature of proteinase K. The experimental data were illustrated inTables as follows.

TABLE 1 DNA amounts from human body according to composition of adhesiveagent in sticker EDTA Tris SDS DNA amount Samples (mol) (mmol) (vol %)(μg/ml) 1 (Control) 0 0 0 0 2 (Example 1, 2) 0.1 10 37.5 45 3 0.05 5 3039 4 0.1 15 40 40 * The DNA amounts were measured by UV absorbance. Forreference, if the absorbance is 1 at 260 nm, dsDNA is estimated to 50μg/ml of concentration.

TABLE 2 DNA amounts from human body according to temperature ofincubation in sticker Samples DNA amount collected (μg/ml) 1 (Control) 02 (37° C.) 45 3 (30° C.) 30 4 (45° C.) 35 * The DNA amounts weremeasured by UV absorbance. For reference, if the absorbance is 1 at 260nm, dsDNA is estimated to 50 μg/ml of concentration.

TABLE 3 DNA amounts from human body according to time period ofincubation in sticker Samples DNA amount collected (μg/ml) 1 (Control) 02 (60 min) 45 3 (40 min) 30 4 (80 min) 35 * The DNA amounts weremeasured by UV absorbance. For reference, if the absorbance is 1 at 260nm, dsDNA is estimated to 50 μg/ml of concentration.

TABLE 4 DNA amounts from human body according to reaction period ofproteinase K (the composition of paint solution is the same with that ofExample 1 and 2) Samples DNA amount collected (μg/ml) 1 (Control) 0 2(90 min) 45 3 (30 min) 20 4 (100 min) 35 * The DNA amounts were measuredby UV absorbance. For reference, if the absorbance is 1 at 260 nm, dsDNAis estimated to 50 μg/ml of concentration.

TABLE 5 DNA amounts from human body according to reaction temperature ofproteinase K (the composition of paint solution is the same with that ofExample 1 and 2) Samples DNA amount collected (μg/ml) 1 (Control) 0 2(65° C.) 45 3 (40° C.) 29 4 (68° C.) 31 * The DNA amounts were measuredby UV absorbance. For reference, if the absorbance is 1 at 260 nm, dsDNAis estimated to 50 μg/ml of concentration.

Experimental Example 2 Comparison of Productive Yields of Human DNAs inthe Sticker of the Present Invention and Conventional Stickers

In order to compare DNA productive yields of the sticker prepared inExamples and conventional stickers, PCR was performed by using ApoE geneand IL-4 gene.

-   -   i) The primer sequences for amplifying ApoE gene were described        as follows:

Forward: 5′-tcggccgcagggcgctgatggac-3′ Reverse:5′-cccaggcgctcgcggatggcgc-3′

-   -   PCR was performed under a following condition.    -   95° C., 5 min; 95° C., 20 sec, 70° C., 1 min, 40 cycles, 72° C.,        1 min; 72° C., 5 min    -   ii) The primer sequences for amplifying IL4 gene were described        as follows:

Forward: 5′-gacccaaactaggcct-3′ Reverse: 5′-cagtcctcctggggaaagat-3′

-   -   PCR was performed under a following condition.    -   95° C., 5 min; 95° C., 30 sec, 62° C., 30 sec, 40 cycles, 72°        C., 1 min; 72° C., 5 min

As illustrated in FIG. 2, conventional stickers and the sticker of thepresent invention varying the composition were used to extract DNAs in 8of target persons. Then, ApoE gene and IL-4 gene were amplified byperforming PCR. As a result, it is identified that both data is improvedin the sticker of the present invention.

Experimental Example 3 Comparison of DNA Productive Yields According toMethods for Separating DNAs from Stickers

In order to compare DNA productive yields of the sticker prepared inExamples according to methods for separating DNAs, PCR was performed byusing ST gene and DRD2 gene.

-   -   i) The primer sequences for amplifying ST gene were described as        follows:

Forward: 5′-ggcgttgccgctctgaatgc-3′ Reverse:5′-gagggactgagctggacaaccac-3′

-   -   PCR was performed under a following condition.    -   95° C., 5 min; 95° C., 20 sec, 60° C., 40 sec, 40 cycles, 72°        C., 1 min; 72° C., 5 min    -   ii) The primer sequences for amplifying DRD2 gene were described        as follows:

Forward: 5′-gatgatacccacttcaggaag-3′ Reverse:5′-gatgtgtaggaattagccagg-3′

-   -   PCR was performed under a following condition.    -   95° C., 5 min; 95° C., 20 sec, 60° C., 40 sec, 40 cycles, 72°        C., 1 min; 72° C., 5 min

As illustrated in FIG. 3, the productive yields of DNAs that areextracted with protein precipitation solution through a traditionalphenol extraction according to the present invention were compared in 5of target persons. ST gene and DRD2 gene were amplified by performingPCR. As a result, it is confirmed that both data is improved, when usingthe extraction method of the present invention.

INDUSTRIAL APPLICABILITY

The sticker for DNA collection and the method for collecting DNA usingthe same enable keratin attached from human skin separated easilywithout blood or hair. Further, the present invention enables a humangene separated conveniently and amplified by using a PCR technique.Therefore, the present invention may attain the effect that identifies areal child, investigates a crime and the like. Hence, the sticker forDNA collection and the method for collecting DNA using the same areindustrially useful in genetic engineering fields.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention.

Those skilled in the art will also appreciate that such equivalentembodiments do not depart from the spirit and scope of the invention asset forth in the appended claims.

1. A sticker for DNA collection, which is covered onto one side with apaint solution comprising 0.05˜1 mol of EDTA (pH 8.0), 0.002˜0.015 molof Tris (pH 8.0), 30˜40 vol % of SDS (approximately 1˜1.4 mol) and 3%peyonine.
 2. A method for collecting DNA using a sticker for DNAcollection, which comprises additional steps: (1) obtaining skin keratinfrom a human body by using the sticker for DNA collection of claim 1;(2) digesting with protease K to separate DNAs from the sticker; andthen, (3) adding a reagent to precipitate proteins in a half volume ofthe resulting solution.